T cell interactions with antigen-presenting cells (APCs) are important both in normal host immune responses and in pathologic conditions including autoimmune disease. Despite their importance, we still have limited understanding of the mechanisms that regulate the formation and release of T cell-APC contacts, critical determinants of T cell activation. The long-term goal of the proposed research is to understand the molecular mechanisms that regulate the duration of T cell-APC contacts and how these 3D adhesive structures affect immune responses and the development of autoimmune disease. Although T cell-APC contact sites, known as the immune synapse, share many structural components with integrin-mediated focal adhesions, we have demonstrated that the adhesive mechanisms that regulate T cell-APC contact site dynamics are distinct. Despite this recent progress, the mechanisms that regulate the formation of transient versus sustained T cell-APC contacts to mediate T cell tolerance or immunity remain largely unknown. Moreover, the kinetics with which components enter and leave T cell-APC contact sites are not well defined. The hypothesis that guides our research is that key regulators of focal adhesion turnover, talin, phosphatidylinositide phosphate kinase PIPKI390 and focal adhesion kinase (FAK) differentially regulate the polarization of the integrin LFA-1 and the dynamic assembly and disassembly of T cell- APC contacts to affect T cell activation and autoimmunity. Substantial evidence suggests that stable T cell-APC interactions are necessary for T cell activation and the development of autoimmune disease. Yet, depletion of LFA-1, which mediates sustained T cell-APC interactions, can lead to worsening autoimmunity in the experimental autoimmune encephalitis (EAE) mouse model of multiple sclerosis, a CD4 T cell driven disease (Gultner, 2010). Therefore, the relationship between adhesive interactions and autoimmunity may be difficult to predict. Using knockout mice we have generated, we will determine how LFA-1 associated proteins, talin, PIPKI390 and FAK regulate the development of autoimmunity in EAE. We are now uniquely positioned to pursue the following specific aims: I. Test the role of talin in T cell-APC interactions and T cell activation. II. Determine how phosphatidylinositide phosphate kinase (PIPKI390) regulates T cell-APC interactions and the development of autoimmunity in EAE. III. Test the role of focal adhesion kinase (FAK) in T cell- APC interactions and T cell activation. The strength of this proposal lies in the use of conditional transgenic mice, use of novel inducible reagents and live 3D time-lapse fluorescent imaging to probe the pathways that regulate the dynamics of T cell-APC contact sites to affect T cell activation. The proposed studies will lead to improved understanding of the mechanisms that regulate T cell-APC interactions and T cell activation and may provide the foundation for new strategies to treat autoimmune disease.